1. Field of the Invention
The present invention is directed to methods and diagnostic kits for providing an objective diagnosis of pain or stress experienced by a patient, and to compositions and methods for the alleviation of pain or stress. The invention further relates to reliable diagnostic and treatment tools useful for indicating the efficacy of pain or stress relieving compositions or methods, and the amount of relief provided by conventional treatments.
2. Description of the Background
Pain is a major aliment affecting the population. The analgesic industry and its advertisements are constant reminders of the magnitude of the problem in the population. Of the many types of medical problems involving pain as a major symptom, chronic spinal pain, with its overwhelming presence in the United States and other countries, is one of the most difficult to treat. It is estimated that primary and secondary expenditures associated with chronic spinal pain averages about $100 billion annually in the United States alone. The collateral loss of private and corporate productivity, while never quantitated, is also expected to be significant.
Health care professionals treating patients with chronic spinal pain recognize the limitations of modern diagnostic methods for assessing chronic spinal pain. Current methods for assessment such as, for example, history and physical examination, questionnaires, x-rays, imaging, electromyelograms, imaging techniques and myelograms all suffer from inherent limitation because of their indirect nature. The prevalence of false positive indicators of pain as well as the rise of pain management industries such as clinics, practitioners and alternative treatment centers, give testimony to the problem and the need for objective, accurate laboratory data.
Accurate assessment of a patient""s pain is a prerequisite to the successful diagnosis and treatment of chronic spinal pain. Without an objective standard, meaningful comparisons of different treatment protocols will rely on the subjective memories of the patient or the health care worker. Age, stress, infirmity and weakness from long illness may affect the patient""s memory. Further, patient self-assessments are of limited value because patients do not always communicate their pain intensity accurately or effectively. Adjectives such as burning, sharp, pressing, stabbing and unbearable are of limited value for comparison between patients. Finally, comparisons of patients with different social, regional, language or cultural background may be extremely difficult because of the choice of adjectives.
In spite of the difficulties in assessment, health care professionals including psychiatrists and psychologists must attempt to adequately assess and manage pain. These attempts require a determination of whether the pain or stress is severe, moderate or mild. A typical diagnosis will also involve a physical examination for accompanying characteristics such as sweating, palpitations, irregular heart beats, fainting sensation, aggravation of pain by deep breathing, pressure, heat or cold. These data, along with any other clinical information, and the patient""s own description, is used to determine the most appropriate treatment.
Such pain assessment systems and treatment are empirical and can only provide a rough estimation of the actual amount of pain. Inaccuracies in the ability to prescribe proper amounts of medication result in an inability to provide proper pain treatment. Prescribing too little medication, i.e. under-medication where an inadequate amount of analgesic is used, results in needless suffering, reduced mobility, prolonged hospital stays and delayed recovery. Using too much medication, i.e. over-medication, can result in increased side effects, possible organ damage, allergic reaction, sleepiness, nausea or chemical dependency on analgesics.
Because the diagnosis of pain is difficult and often, if not usually, inaccurate, the ordinary course of treatment for pain will involve multiple office visits. Each visit will involve feedback from the patient, assessment of the efficacy of treatment and periodical changes in the dosage and the type of medications. Frequent office visits lead to an increase in health care cost and lost productivity, at least in part, due to inadequate treatment of pain (i.e. over-medication and under-medication). Further, if the patient""s condition changes due to an increase or decrease in severity, a new round of initial medication, office visits, feedback and assessment has to be started to manage the pain. An accurate assessment of pain will result in reduced health care costs, with additional benefits such as earlier patient release, earlier mobilization and reduced reliance on hospital and outpatient medical facilities. Thus, given the significance and magnitude of chronic spinal pain, there is a long felt need for a simple, valid and reliable assay, to be used by health care workers to assess a patient""s pain.
Pain is first perceived as a result of the stimulation of specialized nerve endings. The stimulation is transmitted through the nervous system to the brain where the patient perceives the signal as pain. The nervous system, including the brain, comprises about one hundred billion neurons. Each neuron is connected to other neurons in a network. On average, each neuron has, through its axonal and dendritic processes, ten thousand or more connections with other neurons. At the connections of neurons, the cell membranes are not fused but are separated by gaps known as synapses. Signal transduction from neuron to neuron or from neuron to organs (e.g. muscles cells, retina cells, etc.) occur through chemical mediators, referred to as neurotransmitters, that are released into the synapse.
The transmission of a nerve impulse (action potential) along a nerve is electrical and, as such, is measured in millivolts. However, at the synapses, the action potential is transmitted from the pre-synaptic membrane and the post-synaptic membrane of the receiving neuron via protein known as neurotransmitters. The gaps that exist between the neurons and the voltage and current levels of nerve impulses prevent these potentials from passing from one neuron to another neuron directly. Thus, neurotransmitters relay the action potentials between the neurons so nerve impulses can jump this intercellular gap.
When a nerve impulse arrives at the synapse, that impulse is transmitted into a chemical signal via the release of neurotransmitters. The neurotransmitters diffuse rapidly through the intercellular space until it reaches its intended targetxe2x80x94the next neuron or muscle cell. There, the chemical neurotransmitter elicits a response in the recipient cell which induces a reaction such as a nerve impulse or a set of intracellular reactions (without necessarily being accompanied by a change of electrical properties). As a result of this process, a signal that began as a nerve impulse is transmitted from one neuron to another and either enhanced, inhibited or blocked.
About fifty neurotransmitters have now been identified. Some, such as glutamate or acetylcholine stimulate the transmission of nerve impulses and are referred to as excitatory; others, such as [Gamma]-aminobutyric acid (GABA), decrease nerve impulse transmission and are called inhibitory.
GABA, glutamate and acetylcholine (ACh) are the major transmitters of the brain. Evidence has confirmed cholinergic involvement in the antinociceptive effect of GABA (Kendall D. A., et al., J. Pharmacol. Exper. Therapeutics, 220(3):482-7, 1982). Additionally, ACh was thought to be involved in nociception with, or in association with, the endorphinergic and serotonergic systems (Schneck H. J. and Rupreht J., Acta Anaesth. Belg. 40(3):219-28, 1989). There is thought to be a close relationship between cholinergic afferents, substance P interneurons and serotonergic receptors (Feuerstein T. J. et al., Naunyn-Schmiedebergs Archives of Pharmacology, 354(5):618-26, 1996).
The descending connections of the midbrain, especially from the hypothalamus and zona incerta, may be some of the components of the neural networks that regulate nociception (Morrell J. I. et al., J. Comp. Neurol., 201(4):589-620, 1981). A descending or local spinal cholinergic system, together with descending serotonergic and noradrenergic systems, has been found to be involved in the centrifugal inhibition of spinal nociceptive transmission (Zhuo M. and Gebhart G. F., Brain Res., 535(1):67-78, 1990). These cells provided cholinergic innervation to the entire brainstem reticular formation. Investigators have found that ascending fibers to the thalamus and descending fibers into the medullary reticular formation are involved in sensory-motor inhibition (Jones B. E., Neuroscience, 40(3):637-56, 1991).
Another chemical, important in transmission of nerve impulses, is the enzyme serum cholinesterase (SChE). SChE, also known as pseudocholinesterase, has been documented to increase when the neuronal activity of the cholinergic system of the brain is activated with pain such as chronic spinal pain. With this activation, ACh is spilled into extracellular spaces (Kurokawa M. et al., Neuroscience Left., 209(3):181-4, 1996), where it is degraded by SChE (Cooper J. R. et al., The Biological Basis of Neuropharmacology. New York: Oxford University Press, 27-216, 1996; Guyton A. C., Basic Neuroscience, In: Anatomy and Physiology, Philadelphia: W. B. Saunders Co., 1987). ACh is the only neurotransmitter hydrolyzed prior to uptake into the presynaptic neuron for resynthesis; all others are taken up without degradation (Chen D. and Lee K. H., Biochem. Pharmacol., 49(11):1623-31, 1995; Ghelardini C. et al., Life Sc. 58(25):2297-309, 1996). However, some excess, intact ACh is found in the extracellular space. This excess ACh is thought to be degraded by SChE (Cooper J. R. et al., 7 The Biological Basis of Neuropharmacology. New York: Oxford University Press, 27-216, 1996; Kurosawa M. et al., Neurochem. Int. 21(3):423-7, 1992; Scali C. et al., Euro. J. Pharm.; 325(2-3):173-80, 1997). Other investigators report that ACh is degraded primarily in the extracellular space (Todorov L. D. et al., Nature 387:76-9, 1997; Ishii Y. et al., Japanese J. Pharm., 66(3):289-93, 1994). Stimulation of sectioned sciatic nerves in cats also produced a prompt increase of cholinesterase in the cerebral spinal fluid (CSF) (Vogt M. et al., Neuroscience 12:979-995, 1984).
It was reported that noxious stimulation can increase ACh in the cerebral cortex (Mitchell J. F. J. Physiol., 165:98-116, 1963; Phillis J. W. Brain Res., 7:378-9, 1968). However, there are also reports that pain produced an intense neuronal activity (INA) throughout the CNS, and diffuse CNS neuronal activity with pain (Dixon C. E. et al., Neuroscience Lett., 198(2):111-4, 1995; Dubovy P. et al., Cellular Molecular Biol., 36(1):23-40, 1990; Eisenach J. C. et al., Anes. Anal., 82(3):621-6, 1996).
The cholinergic system is part of this neuronal activity. The neurotransmitter ACh was first identified in 1900, and its properties noted in 1925. The ACh system was found widely disposed throughout the CNS and shown to have properties for processing pain. In the synaptic cleft, ACh is degraded into choline and acetate by cholinesterase present in the synaptic area bound to local collagen and glycosaminoglycans. The ACh that is not degraded spills into the extra-cellular space and is degraded by SChE (Cuadra G. and Giacobini E., J. Pharm. Experimental Therapeutics, 275(1):228-36, 1995, Messamore E. et al., Neuropharm. 32(8):745-50, 1993). Injection of physostigmine and heptylphysostigmine into rats appears to result in an elevation of the ACh level in the extracellular space (Cuadra G. and Giacobini E., J. Pharm. Exper. Therapeutics, 275(1):228-36, 1995; Cuadra G. et al., J. Pharm. Exper. Therapeutics, 270(1):277-84, 1994). Further, neostigmine has been shown to inhibit cholinesterase and to produce an extracellular ACh level which is above detection limit (Messamore E. et al., Neuropharm.; 32(8):745-50, 1993). Anti-cholinesterase drugs can increase the extracellular levels of ACh and decrease the level of cholinesterase (Ishii Y. et al., Japanese J. Pharm., 66(3):289-93, 1994). With a turnover time of 150 microseconds, equivalent to hydrolyzing five thousand molecules of ACh per second, cholinesterase ranks as one of the most efficient enzymes (Cooper J. R. et al., The Biological Basis of Neuropharmacology. New York: Oxford University Press, 27-216, 1996).
Stimulation of the nucleus basalis of Meyert increases both cortical blood flow and a release of ACh in the cortex in rats. This stimulation produces a measurable, increase in the extra-cellular ACh (Kurokawa M. et al., Neuroscience Lett., 209(3):181-4, 1996). Further, a stimulus to the paws of anesthetized rats produces a significant (p less than 0.05) elevation of extracellular ACh (Kurosawa M. et al., Neurochem. Int., 21(3):423-7, 1992).
A monitoring system, a periventricular structure, was found in animals to consist of cholinergic receptors in the vessels of the anterior and intermediate pituitary lobes that are known as blood ACh reading bodies (BARBS) (Caffe A. R., Histol. Histopathol., 11 (2):537-51, 1996). When ACh is injected into the brachial artery of humans, extreme pain was produced (Cooper J. R. et al., The Biological Basis of Neuropharmacology. New York: Oxford University Press, 27-216, 1996; Hata T. et al., Japanese J. Pharm. 41(4):475-85, 1986). BARBS may regulate homeostasis of ACh in the blood of the brain. For example, when ACh is high, as would occur with the neuronal activity caused by CSP, BARBS may signal the liver which responds with a homeostatic response to remove excess ACh from plasma by increasing SChE.
Previously, afflictions such as, for example, disease of the kidney or liver, have been shown to correlate with an alteration in the level of cholinesterase. Thus, numerous methods directed to assaying cholinesterase and serum cholinesterase exist (U.S. Pat. Nos. 3,378,463; 3,433,712; 4,271,310; 4,596,772; 4,861,713 and 5,272,061). None of these, however, disclose methods for determining the level of pain perceived by a patient by measuring cholinesterase levels.
The present invention overcomes many of the problems, limitations and disadvantages associated with current strategies and designs and provides novel methods and diagnostic assays for the detection and quantitation of pain and stress.
One embodiment of the invention is directed to methods of diagnosing the intensity of pain perceived by a patient comprising determining the amount of a marker in a biological sample obtained from said patient wherein said marker correlates with the perception of pain. These methods are useful for quantitating and monitoring either acute or chronic pain and especially useful for the detection and measurement of chronic spinal pain. Biological samples in which a marker can be detected include blood, serum, lymph fluid, tears, semen, intracellular fluid, interstitial fluid, cerebrospinal fluid, urine, sweat and saliva. Detection may comprise determination of the amount of marker in the sample or marker may be isolated and purified. Isolation can be performed by electrophoretic separation such as polyacrylamide gel electrophoresis.
Another embodiment of the invention is directed to methods for determining the intensity of a pain perceived by a patient. These methods comprise collecting a biological sample from the patient and that may contain a marker whose presence, absence or quantity correlates with the intensity of pain perceived by a patient. The amount of marker in the sample is determined using, for example, an ELISA or other detection or quantitation tool and the intensity of pain perceived by the patient objectively determined based on the amount of marker in the sample. Preferably the marker is a neurotransmitter or a metabolic product of a neurotransmitter such as cholinesterase. The relative amount of cholinesterase in the sample is determined and compared to the amount of cholinesterase in a control sample obtained from a subject without pain. The patient and the control subject may be the same person or different people or groups of individuals.
Another embodiment of the invention is directed to methods for determining the intensity of a pain perceived by a patient by determining the amount of cholinesterase in a sample of body fluid obtained from the patient. Such methods are particularly useful for the detection and quantitation of chronic pain such as chronic spinal pain.
Another embodiment of the invention is directed to methods for identifying a marker that correlates with the intensity of a pain perceived by a patient. These methods comprise collecting a serum sample from the patient and separating the components of the sample from each other by gel electrophoresis. The gel is reacted with a diazonium salt and a substrate for a period of time to form a detectable band comprising an insoluble diazonium complex. The size and location of the detectable band that correlates with the patient""s perception of pain can be quickly and easily identified.
Another embodiment of the invention is directed to methods for determining the efficacy of a treatment for pain. These methods comprise determining a first severity of pain in the patient by determining the amount of a marker in a first biological sample obtained from the patient. After the desired treatment is administered to the patient, a second severity of pain in the patient is determined by measuring the amount of marker in a second biological sample obtained from the treated patient. By comparing the first severity of pain to the second severity of pain, based on the relative amounts of marker in the samples, an objective assessment of the effectiveness of the treatment can be determined. Such methods may also be used for target validation in determining the most appropriate target in the overall treatment of pain perceived by the patient.
Another embodiment of the invention is directed to diagnostic kits for determining the severity of pain in a patient. These kits comprise at least one agent that reacts with a marker whose presence in a biological sample correlates with the perception pain in a patient from whom the sample is obtained. For example, kits may contain a plurality of antibodies that are specifically reactive against or bind specially to the marker. These antibodies may be polyclonal, monoclonal or simply antibody fragments. The agents may also be substrates when the marker is an enzyme. Substrates that can be used for a cholinesterase marker include ACh and ACh analogs, a protein cleavable by cholinesterase, 4-chloro-2-methylaniline and combinations of these substances.
Another embodiment of the invention is directed to pharmaceutical compositions comprising a therapeuticaly effective amount of a pain-associated marker or an agent that interferes with the perception of pain by the patient. Preferably, the composition selectively inhibits the pain-associated activity of ACh. In a preferred embodiment the pain-associated marker is cholinesterase. Administration of compositions directly or indirectly affect the activity of ACh and interfere with the generation or progression of pain being perceived by the patient.
Another embodiment of the invention is directed to methods of treating a pain being experienced by a patient. Methods of the invention comprise administration of a composition to the patient as determined by the presence of a pain-associated marker in a biological sample obtained from the patient. Compositions may contain new or conventional pharmaceuticals of an amount and type as determined from the presence and quantity of a pain-associated marker in a biological sample obtained from the patient.
Other embodiments and advantages of the invention are set forth, in part, in the description which follows and, in part, will be obvious from this description and may be learned from the practice of the invention.